In Vitro Tests for Assessing the Neutralizing Ability of Snake Antivenoms: Toward the 3Rs Principles

Abstract

There is an urgent need to strengthen the implementation of the 3Rs principle (Replacement, Reduction and Refinement) in the use of experimental animals in toxinological research and in the assessment of the neutralizing efficacy of snake antivenoms. This is a challenging task owing to the inherent complexity of snake venoms. The state of the art on this topic is hereby reviewed, with emphasis on the studies in which a correlation has been observed between in vivo toxicity tests and in vitro surrogate assays, particularly in the study of lethal activity of venoms and its neutralization. Correlations have been described with some venoms-antivenoms when using: (a) enzyme immunoassays, (b) hemagglutination, (c) enzyme assays (proteinase, phospholipase A₂), (d) in vitro coagulant effect on plasma, (e) cell culture assays for cytotoxicity, (f) functional assays for assessing neurotoxicity in vitro, (g) use of hens' eggs, and (h) antivenomics. Additionally, the routine introduction of analgesia in these assays and the design of more 'humane' protocols for the lethality test are being pursued. It is expected that the next years will witness a growing awareness of the relevance of the 3Rs principles in antivenom testing, and that new in vitro alternatives and more 'humane' experimental designs will emerge in this field.

Keywords: 3Rs; analgesia; antivenoms; in vitro assays; lethality assays; neutralization; snake venoms.

Figure 1

Assays included in the WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins for the assessment of antivenom preclinical efficacy. The WHO divides these assays into the ‘essential assay’, which is the analysis of the neutralization of lethal activity of venoms, and ‘additional recommended assays’, which assess the neutralization of other toxic activities, depending on the toxicological profile of the venom under study.

Figure 2

In vitro assay for the assessment of the ability of antivenoms to bind to post-synaptically acting α-neurotoxins from snake venoms. A solution containing a fixed concentration of venom is incubated with various dilutions of antivenom. Then, antibodies (both free and venom-bound) are removed from free low molecular mass toxins (including neurotoxins) by ultrafiltration. The filtrate (containing these toxins) is incubated with purified nicotinic acetylcholine receptor (nAChR). Afterwards, the preparation is added to plate wells that had been coated with a purified α-neurotoxin. Upon incubation and washing, anti-nAChR antibodies are added, followed by washing and addition of conjugated anti-IgG antibodies. After adding the corresponding substrate, the absorbance is recorded. The nAChR preparation, which is obtained from the electric organ of fish, could be substituted by synthetic peptides containing the binding site for α-neurotoxins. For details of this procedure, see Ratanabanangkoon et al. (74).

Figure 3

The 3Rs principles, as applied to the evaluation of the neutralizing ability of antivenoms. The search for Replacement, Reduction and Refinement (3Rs) should be actively pursued in the field of antivenom potency testing. Some examples of the implementation of these principles in antivenom testing are shown.